Estudo da interacção do fungo saprófita Hypholoma fasciculare com microrganismos filamentosos e seu efeito no crescimento de plantas de castanheiro

Hypholoma fasciculare é um fungo saprófita-lenhícola muito comum nos povoamentos de Castanea sativa na região do nordeste transmontano. Nos últimos anos, esta espécie tem sido explorada como agente de controlo biológico, por ser muito combativa contra espécies patogénicas. O presente trabalho tem como objectivo geral avaliar o potencial do H. fasciculare como agente de controlo biológico; e o efeito da sua aplicação nos fungos ectomicorrízicos bem como nas plantas de castanheiro e no processo de micorrização pelo Pisolithus tinctorius. A avaliação do potencial do H. fasciculare como agente de controlo biológico, foi efectuada em condições in vitro pelo método da cultura dupla. O fungo H. fasciculare inibiu significativamente o crescimento dos patogéneos Armillaria mellea e Phytophthora cambivora, e dos fungos micorrízicos Suillus luteus e Pisolithus tinctorius. Os mecanismos adoptados pelo H. fasciculare foram “antagonismo e agonismo à distância”, respectivamente para as espécies patogénicas e micorrízicas. Neste processo parecem intervir enzimas líticas, como a amilase, celulase, lacase e lipase, produzidas pelo H. fasciculare. Observaram-se ainda alterações morfológicas no H. fasciculare possivelmente com o intuito de se tornar mais resistente à invasão da espécie interactuante e/ou mais invasivo. Pelo contrário, nas co-culturas com Amanita gemmata e Colletotrichum acutatum o crescimento de H. fasciculare foi inibido; e com P. cinnamomi e S. bovinus não se observaram diferenças de crescimento para as espécies interactuantes. O efeito da aplicação de H. fasciculare nas plantas de castanheiro e no processo de micorrização pelo P. tinctorius foi avaliada em condições de estufa. As plantas inoculadas com P. tinctorius apresentavam um maior crescimento e níveis foliares de azoto, fósforo, clorofilas (a, b e total) e carotenóides face às plantas não inoculadas. O efeito positivo da micorrização foi contudo suprimido pelo fungo H. fasciculare quando aplicado simultaneamente com P. tinctorius. Este resultado poderá estar relacionado com a inibição da micorrização das raízes pelo H. fasciculare e/ou pela competição entre o P. tinctorius e o H. fasciculare por nutrientes, nomeadamente azoto. O efeito deletério de H. fasciculare não se verificou quando aplicado nas plantas isoladamente ou 30 dias depois da inoculação com P. tinctorius. Os resultados obtidos evidenciam xiv uma interacção antagónica entre fungos ectomicorrízicos e saprófitas com repercussões ao nível do crescimento e fisiologia da planta.Hypholoma fasciculare is a saprophyte-legnicolous fungus very common in communities of Castanea sativa in the northeast region of Portugal. In the last few years this species has been explored as a biological control agent due to its ability to combat pathogenic species. The objective of this study is to evaluate the potential of H. fasciculare to act as a biological control agent and the effects of its application on ectomycorrhizal funguses, chestnut plants as well as in the process of mycorrhization by Pisolithus tinctorius. The evaluation of the potential of H. fasciculare as a biological control agent was made in in vitro conditions and by the double culture method. The H. fasciculare fungus inhibited significantly the growth of the pathogens Armillaria mellea and Phytophthora cambivora and the micorrizic Suillus luteus and Pisolithus tinctorius. The mechanisms adopted by H. fasciculare to act on pathogenic species and micorrizic species were respectively “antagonism and agonism at a distance”. In this process it is observed the intervention of litic enzymes such as amylase, cellulase, lacase and lipase, all produced by H. fasciculare. It is also observed morphological alterations of H. fasciculare possibly with the intent of becoming more resistant to the possible invasion of the interacting and/or more invasive species. On the other hand, in the co-cultures with Amanita gemmata and Colletotrichum acutatum, the growth of H. fasciculare was inhibited. With P. cinnamomi and S. bovinus no differences of growth was observed by the interacting species. The effects of the application of H. fasciculare in the chestnut plants and in the process of micorrization by Pisolithus tinctorius was evaluated in greenhouse conditions. The plants inoculated with P. tinctorius showed a greater growth, at a foliar level, of nitrogen, phosphorous, chlorophylls (a, b and total) and caratanoids relatively to the non-inoculated plants. The positive effect of the micorrization was however suppressed by the H. fasciculare fungus when applied simultaneously with P. tinctorius. This result may be related to the inhibition of the micorrization of the roots by H. fasciculare and/or by the competition between P. tinctorius and H. fasciculare for nutrients, namely nitrogen. The deleterian effect of H. fasciculare was not observed when applied to plants in isolation neither after 30 days of inoculation by P. tinctorius. xvi The results obtained show an antagonic interaction between ectomicorrizic and saprophyte funguses with repercussions in the growth and the physiology of the plant

Genetic diversity of the Chestnut blight fungus Cryphonectria parasitica and its associated hypovirus in Portugal

The European chestnut (Castanea sativa Mill.) agro‐ecosystem has been of high social, economic, and landscape importance in Portugal. Chestnut blight caused by the fungus Cryphonectria parasitica is considered a major cause of the decline of chestnut trees across Europe. C. parasitica is an ascomycete (Diaporthales) that is native to eastern Asia. Infection of chestnut trees with this pathogen is typically associated with extensive bark necrosis (so‐called cankers) on stems and branches, resulting in the subsequent death of the part of the tree above the infection point. Chestnut blight in Portugal was first reported in 1992 and since expanding in distribution. Here, we investigated the invasion history of C. parasitica and its associated hypovirus in Portugal. For this, we characterized 137 isolates collected between 2013 and 2014 in four chestnut stands for virus‐infection, vegetative compatibility (vc) type, mating type and microsatellite haplotype. A total of 33 haplotypes and four vc types were observed, although the Portuguese C. parasitica population is currently dominated by a single haplotype and a single vc type (EU‐11). Further diversification may be expected due to ongoing sexual recombination, but eventually also to new migration and additional introductions. Cryphonectria hypovirus 1 (CHV‐1) was found in two populations. Genetic analysis of the six CHV‐1 isolates obtained revealed that three viral strains belong to the Italian subtype and three to the French subtype, which suggest different, independent introductions.info:eu-repo/semantics/publishedVersio

Biological control of chestnut blight. Detection, identification and characterization of the Hypovirus - CHV1

O cancro do castanheiro é provocado pelo fungo Cryphonectria parasitica é considerado a principal causa do declínio dos castanheiros na Europa. C. parasitica é um ascomiceta (Diaporthales) nativo do continente asiático. A infeção do castanheiro manifesta-se pelo aparecimento de necroses extensas na casca dos ramos e troncos, que resulta na morte dos castanheiros. A hipovirulência é um método seletivo de controlo biológico do Cancro do Castanheiro, trata-se de uma infeção do fungo C. parasitica com Cryphonectria hypovirus 1 (CHV1), um vírus de dsRNA não encapsulado do género Hypovirus. O controlo biológico com estirpes hipovirulentas é considerado um método eficaz no controlo da doença e promove a recuperação dos castanheiros. A aplicação necessita da produção em laboratório de estirpes hipovirulentas compatíveis com a estirpe virulenta presente no campo e a sua introdução na margem de cancros ativos. Neste estudo pretendeu-se detetar, identificar e caracterizar o hipovirus CHV1 com potencial para integrar programas de controlo biológico do Cancro do castanheiro em Portugal. Com a utilização de métodos moleculares foram identificados e caracterizados 11 hipovirus em isolados brancos de C. parasitica obtidos em cancros no nordeste de Portugal.The chestnut blight caused by the fungus Cryphonectria parasitica is considered a major cause of the decline and disappearance of chestnut trees across Europe. C. parasitica is an ascomycete fungus (Diaporthales) that is native to eastern Asia. Infection of chestnut plants with this fungus is typically associated with extensive necrosis (cankers) of the bark on stems and branches, resulting in the subsequent death of the tree. Hypovirulence is a specific method for biological control of Chestnut Blight, it is an infection of C. parasitica with Cryphonectria hypovirus 1 (CHV1), a unencapsidate dsRNA virus of the genus Hypovirus. Biological control with hypovirulent strains is considered an efficient method to control the disease and improve the host plant recovery. The field application needs laboratorial production of compatible hypovirulent strain and introduction by inoculation at the margin of active cankers. In this work it was intended to identify and characterize the hypovirus with potential ability to integrate biological control programmes against Chestnut Blight. Using molecular techniques 11 hypovirus with hypovirulence potential were identified on white isolates of C. parasitica isolated on cankers from northeast of Portugal

Entomopathogenic fungi associated with the main insect pest in the Northeast of Portugal: preliminary results

Due to the problems caused by the use of chemical insecticides for humans and environment alternative pest control methods are an important topic of research. The use of microbial insecticides especially fungal agents are an attractive and promising alternative for biological control of insect pests. The aim of this work was to identify naturally occurring entomophatogenic fungi on the olive moth, Prays oleae Bern., in the northeast of Portugal, as first step to select biological control agent again this olive pest. The experimental work was carried out during 2007 in the three generation of the insect (phyllophagous, anthophagous and carpophagous generation). In each generation P. oleae larvae and pupae were collected in different groves and were put in glass vials in a climatic chamber with a photoperiod of 12h light:12h dark, 22ºC (light): 16ºC (dark) and 60% relative humidity, until emergency of the adults. From dead larvae, fungi were isolated on PDA plates and incubated at room temperature. Pure cultures were morphological and molecularly identified based on the ITS region of the rDNA. From the identified species Beauveria bassiana Vuill. and Cordyceps sinensis (Berk.) Sacc. were the most promising being B. bassiana the most abundant one.This work was partially financed by the project INTERREG III

Natural spread of hypovirulence in Cryphonectria parasitica. A case study, Sergude - Minho – Portugal

El castaño (Castanea sativa) es una especie de gran importancia económica y social en la región del Norte de Portugal. Uno de los mayores obstáculos en el aumento de la producción de castaña está relacionada con la presencia de factores bióticos como la antigua y conocida enfermedad de la Tinta y más recientemente con el chancro que provoca elevada mortalidad en los castaños. No hay sustancias químicas capaces de bloquear el desarrollo del chancro del castaño y todos los medios de lucha cultural mostraron baja eficacia. La hipovirulencia es el medio de lucha biológica y la manera más ventajosa para parar la enfermedad en Europa. El método mostro una elevada capacidad de cura en los chancros y la recuperación completa de los castaños atacados. El mecanismo molecular de hipovirulencia está asociado a la presencia en el hongo parasítico del virus dsRNA denominado Hypovirus (CHV). En Sergude (Minho) en una parcela con todos los árboles atacados por C. parasitica se verifico en un periodo de 3 – 4 años la remisión natural de los chancros llevando a la recuperación de los árboles enfermos. Conocer los mecanismos implicados en la expresión de la hipovirulencia y los factores asociados a su dispersión natural fue el objeto de este trabajo. Se estudió la estructura poblacional de C. parasitica presente en la parcela así como la micoflora epifítica y endofítica asociada.Chestnut (Castanea sativa) is a species of great economic and social importance in the Northern region of Portugal. One of the biggest barriers to increased nut production is related to the presence of biotic factors such as the old and familiar Ink Disease and more recently the Chestnut Blight that is causing high mortality in chestnut. In the absence of chemical fungicides capable of stopping the development of the Chestnut Blight and considering the reduced effectiveness of other mitigation measures, hypovirulence is the most advantageous way to control the disease in Europe. The method showed high ability to heal the cankers and promote the complete recovery of chestnut. The molecular mechanism of hypovirulence is associated with the presence of dsRNA virus of the genus Hypovirus. In Sergude (Minho) in a chestnut grove with all trees attacked by C. parasitica in a period of 3-4 years occurred the natural remission of cankers leading to the recovery of diseased trees. Our objective was to understand the mechanisms involved in the expression of hypovirulence and factors associated with its natural dispersion . We studied the population structure of C. parasitica present in chestnut and associated epiphytic and endophytic mycoflor

INCOME SMOOTHING AND CORPORATE GOVERNANCE

Income smoothing is a strategy aimed at altering accounting results in order to reduce fluctuations. In the other hand, by adopting corporate governance mechanisms it may be possible to reduce information asymmetry and, consequently, reduce the possibility of smoothing results. The general objective of the present study was to investigate the relationship between the adoption of corporate governance mechanisms and income smoothing. Financial data were selected from 211 Brazilian public companies between 2000 and 2015. The firms were separated according to whether or not they adhered to B3’s differentiated corporate governance segments. Income smoothing was measured using both the model by Eckel (1981) and a version of the model proposed by Leuz et al., (2003). The analysis was performed using non-parametric Wilcoxon rank sum tests and descriptive analyses. Based on the descriptive analysis, it was not possible to state that the group of firms adhering to the corporate governance levels had a smaller proportion of companies that smooth results; and the hypothesis that the degree of income smoothing is lower for such companies was not confirmed. The assumption that levels of corporate governance reduce the possibility of income smoothing was not validated based on non-parametric tests. Based on the descriptive analysis, it was concluded that both the proportion of companies that smooth results, and the degree of smoothing by organizations following the B3’s corporate governance levels are not lower than the ones shown by companies that do not follow the levels

Caracterização da estrutura populacional de Cryphonectria parasitica para aplicação da luta biológica por hipovirulência: um caso de estudo na Serra da Padrela

O castanheiro europeu (Castanea sativa Mill) é a principal espécie do sistema agroflorestal nas regiões montanhosas de Portugal. O castanheiro é atualmente afetado por vários agentes patogénicos agressivos, destacando-se o fungo Cryphonectria parasitica, que causa o Cancro do Castanheiro, que provoca a morte da árvore. A Hipovirulência, associada à presença de Cryphonectria hypovirus 1 no fungo fitoparasita, é um método específico e eficiente no controlo biológico do Cancro do Castanheiro. Para uma aplicação eficaz deste método no campo é necessário conhecer as características das estirpes agressivas de C. parasitica presentes no campo. Na Serra da Padrela foi selecionado um souto para realizar este estudo. Foi efetuado uma caracterização dos cancros ativos, e posteriormente o seu tratamento por Hipovirulência. Dos isolados obtidos dos cancros, 99% eram agressivos, sendo identificados 3 isolados como hipovirulentos. Todos foram classificados como vc-type EU-11, estando em maior abundância o mating-type 2, numa proporção de 2:1 comparativamente ao mating-type 1. Os cancros agressivos foram tratados com as estirpes hipovirulentas compatíveis tendo-se observado ao fim de 1 ano uma recuperação das árvores afetadas tendo-se confirmado a presença das estirpes hipovirulentas em cancro anteriormente agressivos

Eficácia do tratamento do Cancro do Castanheiro em ensaios de inoculação com estirpes hipovirulentas de Cryphonectriaparasitica(CHV1) em Trás-os-Montes (Portugal)

O castanheiro europeu (Castanea sativa Mill) é a principal espécie do sistema agroflorestal nas regiões montanhosas de Portugal. O castanheiro é atualmente afetado por vários agentes patogénicos agressivos, destacando-se o fungo Cryphonectria parasitica, que causa o Cancro do Castanheiro, que provoca a morte da árvore. A Hipovirulência, associada à presença de Cryphonectria hypovirus 1 no fungo fitoparasita, é um método específico e eficiente no controlo biológico do Cancro do Castanheiro. Para uma aplicação eficaz deste método no campo é necessário conhecer as características das estirpes agressivas de C. parasitica presentes no campo. Na Serra da Padrela foi selecionado um souto para realizar este estudo. Foi efetuado uma caracterização dos cancros ativos, e posteriormente o seu tratamento por Hipovirulência. Dos isolados obtidos dos cancros, 99% eram agressivos, sendo identificados 3 isolados como hipovirulentos. Todos foram classificados como vc-type EU-11, estando em maior abundância o mating-type 2, numa proporção de 2:1 comparativamente ao mating-type 1. Os cancros agressivos foram tratados com as estirpes hipovirulentas compatíveis tendo-se observado ao fim de 1 ano uma recuperação das árvores afetadas tendo-se confirmado a presença das estirpes hipovirulentas em cancro anteriormente agressivos

Determination of amatoxins and phallotoxins in Amanita phalloides mushrooms from northeastern Portugal by HPLC-DAD-MS

Amanita phalloides is a toxic mushroom responsible for the majority of deaths occurring after mushrooms ingestion, mainly due to amatoxins. In the present study the contents and distribution of the major amatoxins and phallotoxins in different tissues of A. phalloides from two different sites of Portugal were analyzed by liquid chromatography (LC) coupled to diode array (DAD) and mass spectrometry (MS) detection. The main toxins were separated by LC and its chemical structures confirmed by MS. a-Amanitin contents in caps, stipe and volva tissues were quantified by RP-HPLC. The results show that caps have the highest content of amatoxins, whereas the volva was richest in phallotoxins. Moreover variability in the toxins composition from different geographic sites was also observed. This study provides for the first time the content of toxins in A. phalloides from Portugal.Authors are grateful to Dr Zélia dos Santos Azevedo, Faculty of Sciences, University of Porto, who loaned LC/DAD-ESI/MS and for all technical assistance. The authors also are grateful for the help of the Foundation for the Science and Technology (FCT, Portugal) for financial support and also thank FCT for doctoral grant SFRH/BD/74979/2010.info:eu-repo/semantics/publishedVersio

Identificação de bactérias em ambulâncias: riscos assistenciais à segurança do paciente

The aim was to identify microorganisms through the microbial cultivation of bacteria inside ambulances in two municipalities in Baixada Fluminense, in the State of Rio de Janeiro, and to test the sensitivity of these microorganisms to antimicrobials. Samples were obtained from nine specific points inside 6 ambulances. They were inoculated onto salt and blood mannitol agar and were analyzed using matrix-assisted laser desorption and ionization techniques. The sensitivity of seven samples was tested using the disk diffusion method on Muller-Hinton agar. Of the fifty-two samples collected, thirty-one (59.6%) were positive for some genus of bacteria, two (3.80%) were not recognized by MALDI and nineteen (36.6%) were negative. Infection prevention and control practices need to be reinforced, as contamination can result in possible complications for patients.Objetivou-se identificar microrganismos por meio do cultivo microbiano de bactérias do interior de ambulâncias em dois municípios da Baixada Fluminense, no Estado do Rio de Janeiro, e testar a sensibilidade desses microrganismos a antimicrobianos. Amostras foram obtidas de nove pontos específicos do interior de 6 ambulâncias. Elas foram inoculadas no ágar manitol salgado e de sangue, e foram analisadas por meio de técnica de ionização e dessorção a laser assistida por matriz. Foi testada a sensibilidade de sete amostras com o método de difusão em disco em ágar Muller-Hinton. Das cinquenta e duas amostras coletadas, trinta e uma (59,6%) positivaram para algum gênero de bactéria, duas (3,80%) não foram reconhecidas pelo MALDI e dezenove (36,6%) negativaram. É preciso que sejam reforçadas as práticas de prevenção e controle de infecções, dado que a contaminação pode resultar em possíveis complicações para os pacientes